Defructosylation method

ABSTRACT

The present invention is directed to a defructosylation enzyme originating from a plant, a method of defructosylating a fructosylated peptide or protein through use of the enzyme, and a method of measuring a fructosylated peptide or protein.

TECHNICAL FIELD

The present invention relates to a method of defructosylating afructosylated peptide or a fructosylated protein through use of anenzyme, to a novel enzyme having defructosylating ability, and to amethod of assaying a fructosylated peptide or a fructosylated proteinthrough measuring a reaction product obtained from the defructosylationmethod.

BACKGROUND ART

Hemoglobin (Hb) Alc is a stable Amadori product formed through Amadorirearrangement of a Schiff base which is nonenzymatically producedbetween the amino group of β-chain N-terminus valine and the aldehydegroup of glucose. It is also classified as a glycated protein having astructure formed of a valine residue and fructose bonded thereto.Clinically, HbAlc is correlated with a mean blood sugar level of pastone to two months, and therefore, HbAlc serves as an important indicatorin control of diabetes. Thus, there still exists demand for aquantitative HbAlc assay method which is rapid, convenient, accurate,and practical.

As a practical standard assay methodology for HbAlc, IFCC (InternationalFederation of Clinical Chemistry and Laboratory Medicine) adopts amethod which includes separation, by HPLC, of a β-N-terminal 6-peptidefragment which is likely to have fructosyl valine and is obtainedthrough hydrolysis of hemoglobin with endoprotease Glu-C andquantitation of the separated fragment through capillary electrophoresisor mass spectrometry (Kobold U., et al; Candidate Reference Methods forHemoglobin Alc Based on Peptide Mapping; Clin. Chem., 43, 1944-1951(1997)). However, this method requires a special apparatus and entailscumbersome maneuvers and poor economy, making this method impractical.

Existing methods for measuring HbAlc which are currently employed inpractice include HPLC employing, as a carrier, a special hard gel havinga hydrophobic group or a cation exchange group, and lateximmunoagglutination employing anti-HbAlc antibody. These existingmethods, requiring expensive instruments or multi-step immunologicalreactions, are not necessarily satisfactory in terms of speed,convenience, or accuracy.

In recent years, there have been reported enzyme-based assay method forglycated proteins, such as HbAlc and glycated albumin, which includedegradation of glycated protein with protease, and employment offructosyl amino acid oxidase (FAOD) or a similar enzyme that reacts on aglycated amino acid (Japanese Patent Application Laid-Open (kokai) Nos.H05-192193, H07-289253, H08-154672, H06-046846, and H08-336386,WO97/13872, WO02/06519, Japanese Patent Application Laid-Open (kokai)No. 2001-054398).

In any of these methods, in order to avoid difficulty encountered byFAOD or a similar enzyme in acting on glycated protein, regardless ofthe glycated protein being HbAlc or glycated albumin, glycated aminoacids (fructosyl valine for HbAlc; fructosyl lysine for glycatedalbumin) which are characteristic to respective glycated proteins arecut out from a glycated peptide or glycated protein, and the obtainedglycated amino acids are used as substrates for FAOD, etc. Therefore,glycated amino acids for such purposes have to be cut out effectively sothat they can serve as substrate for FAOD, etc.

To achieve the above object, research efforts have been undertaken tosearch for a protease which enables glycated amino acids to beeffectively cut out from glycated protein, and heretofore, numerousproteases have been reported. However, no report has disclosedinformation about the method of cutting out the glycated amino acid (ora peptide containing the glycated amino acid) from a glycated protein,or, in more specifically, the length of the peptide chain cut out fromthe glycated protein. From this viewpoint, therefore, it remains unknownas to whether or not the disclosures of the above publications are infact practical.

Meanwhile, Japanese Patent Application Laid-Open (kokai) No. 2001-95598discloses a method for measuring glycated protein, in which a sample istreated with protease, and the resultant free-form glycated peptide isreacted with glycated peptide oxidase. In the disclosed method, however,there still remains an unsolved problem in that, since the glycatedpeptide oxidase substantially acts on fructosyl dipeptide, the method isnot effective for a fructosyl peptide, which is longer than fructosyldipeptide, and similar to the case of the conventional approach of usingFAOD or a similar substance, a fructosyl dipeptide capable of serving asa substrate must be cut out effectively.

In another report, FAOD is used in combination with another enzyme(Japanese Patent Application Laid-Open (kokai) No. 2000-333696).However, the disclosed method is directed to improvement in measurementsensitivity by measuring hydrogen peroxide from two different sources;i.e., hydrogen peroxide generated from reaction between FAOD andglycated amino acid cut out with protease, and the other hydrogenperoxide generated from reaction between glucosone, which is aconcurrently generated degradation product of glycated amino acid, andglucose oxidase. Thus, the method does not intend to performdefructosylation of glycated peptides of different lengths.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide an enzymeexhibiting defructosylation action on HbAlc or other fructosylatedproteins, or fructosylated peptides of different sizes obtained throughcutting such fructosylated proteins; a method of defructosylation by useof the enzyme; and a method of measuring fructosylated peptide orprotein making use of a defructosylation reaction.

The present inventors have devoted efforts to attain the above object bysearching for a useful enzyme in the natural world. As a result, theyhave found that, as contrasted to the fact that existing enzymes, suchas FAOD, which have been reported to be endowed with defructosylationaction are derived from microorganisms, certain plant families such asRosaceae, Vitaceae, and Umbelliferae are sources of enzymes exhibitingdefructosylation action, and that enzymes originating from such plantsexhibit defructosylation action, regardless of the length of the peptidechain of a fructosyl peptide, thereby leading to completion of theinvention.

Accordingly, the present invention provides a method fordefructosylating a fructosylated peptide or protein, characterized bycomprising reacting, with the peptide or protein, an enzyme which isextracted from a plant and exhibits defructosylation action.

The present invention also provides an enzyme having defructosylating afructosylated peptide or protein, the enzyme being extracted from aplant.

The present invention also-provides a method for measuring afructosylated peptide or protein, characterized by comprising measuringat least one reaction product obtained through use of the abovedefructosylation method.

The defructosylation enzyme of the present invention enablesdefructosylation of N-terminal-valine fructosylated peptide or protein.Moreover, through quantitation of the resultant reaction product, asimilar substance of N-terminal-valine fructosylated peptide, protein,or subunits of protein, e.g. HbAlc, can be quantitatively determinedaccurately.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of capillary electrophoresis obtained from thereaction mixture 1 prepared by reacting a Rosaceae-plant-origindefructosylation enzyme with fructosyl dipeptide (f-VH).

FIG. 2 shows the results of capillary electrophoresis obtained from thecontrol solution 1 prepared by reacting purified water with fructosyldipeptide (f-VH).

FIG. 3 shows the results of capillary electrophoresis obtained from thereaction mixture 2 prepared by reacting a Rosaceae-plant-origindefructosylation enzyme with fructosyl tripeptide (f-VHL).

FIG. 4 shows the results of capillary electrophoresis obtained from thecontrol solution 2 prepared by reacting purified water with fructosyltripeptide (f-VHL).

FIG. 5 shows the results of capillary electrophoresis obtained from thereaction mixture 3 prepared by reacting a Rosaceae-plant-origindefructosylation enzyme with fructosyl tetrapeptide (f-VHLT).

FIG. 6 shows the results of capillary electrophoresis obtained from thecontrol solution 3 prepared by reacting purified water with fructosyltetrapeptide (f-VHLT).

FIG. 7 shows the results of capillary electrophoresis obtained from thereaction mixture 4 prepared by reacting a Rosaceae-plant-origindefructosylation enzyme with fructosyl pentapeptide (f-VHLTP).

FIG. 8 shows the results of capillary electrophoresis obtained from thecontrol solution 4 prepared by reacting purified water with fructosylpentapeptide (f-VHLTP).

FIG. 9 shows the results of capillary electrophoresis obtained from thereaction mixture 5 prepared by reacting a Rosaceae-plant-origindefructosylation enzyme with fructosyl hexapeptide (f-VHLTPE).

FIG. 10 shows the results of capillary electrophoresis obtained from thecontrol solution 5 prepared by reacting purified water with fructosylhexapeptide (f-VHLTPE).

FIG. 11 shows the results of capillary electrophoresis obtained from thereaction mixture 6 prepared by reacting a Vitaceae-plant-origindefructosylation enzyme with fructosyl dipeptide (f-VH).

FIG. 12 shows the results of capillary electrophoresis obtained from thecontrol solution 6 prepared by reacting purified water with fructosyldipeptide (f-VH).

FIG. 13 shows the results of capillary electrophoresis obtained from thereaction mixture 7 prepared by reacting an Umbelliferae-plant-origindefructosylation enzyme with fructosyl dipeptide (f-VH).

BEST MODE FOR CARRYING OUT THE INVENTION

As used herein, the term “defructosylation” refers to a chemical processin which a fructosyl moiety of fructosyl amino acid or fructosyl peptide(i.e., fructosylated amino acid or fructosylated peptide) undergoesoxidation decomposition, hydrolysis, or a similar reaction, to therebygenerate non-fructosylated amino acid or peptide.

No particular limitation is imposed on the enzyme employed in thepresent invention (hereinafter referred to as “defructosylationenzyme”), so long as the enzyme exhibits defructosylation action on afructosylated peptide or protein. However, defructosylation enzymesoriginating from a plant are preferred, since such enzymes are capableof acting on fructosyl peptides having different lengths. No particularlimitation is imposed on the plant containing the enzyme of the presentinvention. However, plants belonging to the family Rosaceae, Vitaceae,or Umbelliferae are particularly preferred. Examples of the plantsbelonging to the family Rosaceae include Malus, Pyrus pyrifolia, Prunuspersica, and Prunus mume. Examples of the plants belonging to the familyVitaceae include Vitis vinifera and Parthenocissus tricuspidata.Examples of the plants belonging to the family Umbelliferae includeDaucus carota, Oenanthe javanica, and Cryptotaenia japonica. Noparticular limitation is imposed on the portion of a plant employed forextracting the enzyme of the present invention, so long as the portioncontains the defructosylation enzyme, and portions such as fruit, leaf,stem, flower, rhizome, and root may be employed. Alternatively, productsof these plants such as juice produced from extracts and freeze-driedpreparations may also be employed.

Extraction of the defructosylation enzyme from such a plant may beperformed by directly disrupting and then squeezing the plant.Alternatively, prior to disruption and extraction, an appropriate bufferor a similar solution may be added to the plant. In the presentinvention, an extract as such may be employed. However, a purifiedextract is preferred. Purification may be performed through a knownmethod. Specifically, there may be employed a suitable combination ofany of ammonia sulfate fractionation processes and column chromatographyprocesses such as ion-exchange chromatography, hydrophobicchromatography, hydroxyapatite gel, and gel filtration. In order toremove the effect of polyphenol contained in the plant extract,additional treatments may be performed through use of a reducing agent,absorbent polymer, or a similar agent.

The defructosylation enzyme of the present invention enables effectivemeasurement of glycated protein, since the defructosylation enzyme ofthe present invention is capable of acting on fructosyl peptides of anysize that are produced from glycated protein through proteasedecomposition, thus eliminating need to add another protease forcleaving the protein and time for the treatment. In addition, thedefructosylation enzyme of the present invention finds utility not onlyin clinical tests, but also in various other fields, including themedical field. The defructosylation enzyme of the present invention mayexhibit, similar to FAOD, decomposition action on the substratefructosyl peptide through oxidation, to thereby defructosylate thepeptide while generating hydrogen peroxide, glucosone, or othersubstances. The defructosylation enzyme of the present invention havingsuch action is particularly preferred, since the generated hydrogenperoxide can be measured in a known measurement system through use of anenzyme such as peroxidase. Alternatively, there may be employed anenzyme which is capable of attaining defructosylation of a fructosylpeptide through hydrolysis while generating glucose, which can bemeasured through use of, for example, glucose oxidase.

No particular limitation is imposed on the fructosylated peptide orfructosylated protein to be treated through the defructosylation methodof the present invention, so long as the defructosylation enzyme acts onthe peptide or protein. However, HbAlc and fructosyl peptides havingfructosylated valine at the N-terminus of a hemoglobin β-chain areparticularly preferred. No particular limitation is imposed on thenumber of amino acid residues contained in the N-terminal-valinefructosylated peptide. However, fructosyl peptides having an amino acidsequence represented by any of SEQ ID NOs: 1 to 5 are particularlypreferred.

The above-described N-terminal-valine fructosylated peptide may beprepared by treating a peptide or protein having any of theabove-mentioned sequences such as HbAlc with, for example, a suitableendoprotease or exoprotease. Examples of the protease include elastase,proteinase K, pepsin, alkaline protease, trypsin, proline-specificendoprotease, V8 protease, carboxypeptidase A, and carboxypeptidase B.The protease used to prepare the fructosyl peptide preferably exhibitsan activity of 0.05 to 10,000 U/mL, more preferably 10 to 2,000 U/mL.

Among a variety of conditions under which the defructosylation enzyme ofthe present invention is reacted with a fructosylated peptide orprotein, the treatment temperature is preferably 20 to 50° C., morepreferably 30 to 40° C., and the treatment time is preferably 3 minutesto 100 hours, more preferably 5 minutes to 20 hours. Through thetreatment, reaction products containing glucosone or glucose anddefructosyl peptides are obtained. Therefore, through measuring one ormore of the reaction products, fructosylated peptide or protein can bemeasured.

In order to determine the enzyme activity of the defructosylation enzymeof the present invention or to determine a fructosylated peptide orprotein, generated defructosyl peptides may be isolated and identifiedthrough HPLC or capillary electrophoresis. Alternatively, an appropriatecarboxypeptidase may be reacted with the defructosyl peptide, to therebydetect or measure the generated amino acid residues. For example, whenthe fructosyl peptide having fructosylated valine at the N-terminusthereof and having an amino acid sequence represented by SEQ ID NO: 5 isreacted with carboxypeptidase, glutamic acid (Glu), proline (Pro),threonine (Thr), leucine (Leu), histidine (His), and valine (Val) aregenerated. Among these amino acid residues, Glu, Leu, and Val can bedetected or determined by measuring the amount of NADH or NADPH producedthrough use of glutamate dehydrogenase, leucine dehydrogenase, andvaline dehydrogenase, respectively. Glucosone or glucose producedthrough defructosylation can be detected or determined by generatinghydrogen peroxide through use of, among other enzymes, glucose oxidaseand then measuring the produced hydrogen peroxide in a peroxidasecolor-developing system. This process is exemplified as follows.

-   (Measurement of product of enzyme reaction)

The enzyme of the present invention is reacted with a substratefructosyl peptide, and the reaction mixture is heated for apredetermined period of time. To the reaction mixture (300 μL), asolution mixture which has been prepared in advance by mixing 200 mMacetate buffer (pH 6.0) (750 μL), 4,000-u/mL glucose oxidase (ToyoboCo., Ltd.) (450 μL), 0.15% 4-aminoantipyrine (300 μL), 0.3% TOOS(Dojindo Laboratories) (300 μL), 500-u/mL peroxidase (Toyobo Co., Ltd.:Type III) (300 μL), and 1% sodium azide (300 μL) is added, and theresultant mixture is treated for 10 minutes at 37° C., followed bymeasurement of absorbance at 550 nm. The above procedure is repeated,except that the substrate is replaced by purified water (control). Theamount of the enzyme reaction product (glucosone or glucose) iscalculated from the developed color through use of a calibration curveprepared by performing the same procedure in which serial dilutions ofglucose are employed instead of the substrate and purified water isemployed instead of the enzyme of the present invention. When adefructosylation enzyme which produces hydrogen peroxide throughdefructosylation reaction is employed, generated hydrogen peroxide canbe directly detected or determined in a known peroxidasecolor-developing system.

No particular limitation is imposed on the peroxidase (POD)color-developing system. Suitable is a method in which a chromogen andPOD are added to the reaction system so that the chromogen is oxidized,thereby producing a color-developing substance, followed by measuringthe substance. As the chromogen, there may be employed a combination of4-aminoantipyrine and a phenol compound, a naphthol compound, or ananiline compound, a combination of MBTH (3-methyl-2-benzothiazolinonehydrazone) and an aniline compound, leucomethylene blue, or similarsubstances. Alternatively, a method as described in Japanese Patent No.2516381 may be employed. Specifically, in the presence of POD, hydrogenperoxide is reacted with divalent cobalt ion, and the produced trivalentcobalt ion is treated with a trivalent-cobalt-ion-specific indicatorsuch as TASBB (2-(2-thiazolyazo)-5-disulfobutylaminobenzoic acidtrisodium salt), thereby producing a color-developing chelate compound,followed by measuring the chelate compound. The latter method providesmeasurement sensitivity 5 to 10 times that provided by the formermethod. As an alternative reagent for detecting hydrogen peroxide,TPM-PS(N,N,N′,N′,N″,N″-hexa(3-sulfopropyl)-4,4′,4″-triaminotriphenylmethane)(product of Dojindo Laboratories), which can be measured with highsensitivity, or a similar reagent may be employed.

According to the method of the present invention, a peptide or proteinhaving a fructosyl valine residue at the N-terminus thereof such asHbAlc can be quantified with very high accuracy. Examples of testsamples to be employed to quantify HbAlc include whole blood anderythrocyte.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1

Preparation of defructosylation enzyme originating from a plantbelonging to the family Rosaceae

The skin and core portion including seeds of a Pyrus pyrifolia fruitwere removed, and, to the remaining flesh portion, 100 mM sodium acetate(pH 4.5) (100 mL/100 g of the flesh portion) containing 300 mM sodiumchloride was added, and the resultant mixture was directly crushed bymeans of a mixer. The product was subjected to centrifugal separation toremove solid substances, whereby a crude extract was prepared. To thecrude extract, PVPP (polyvinylpolypyrrolidone: product of NacalaiTesque, Inc.) was added in an amount of 2%, followed by stirring for 30minutes at room temperature. Thereafter, PVPP was removed throughcentrifugal separation, to thereby yield a treated solution, whichserved as a crude enzyme solution.

Example 2

Preparation of defructosylation enzyme originating from a plantbelonging to the family Vitaceae

The skin of a Vitis vinifera fruit was removed, and the remaining fleshportion was crushed by means of a mixer. The product was subjected tocentrifugal separation to remove solid substances, whereby a crudeextract was produced, which served as a crude enzyme solution.

Example 3

Preparation of defructosylation enzyme originating from a plantbelonging to the family Umbelliferae

A Daucus carota rhizome was directly disrupted through use of a juicer,followed by centrifugation, to thereby remove solid substances. Thethus-prepared crude extract was filtrated through use of a Millex filter(0.45 μm: product of Millipore Corporation), whereby a transparentextract was prepared. Cold ethanol (4 mL) was added to the extract (3mL), and the formed precipitate was removed through centrifugation. Tothe supernatant, cold ethanol was added again, and the formedprecipitate was collected through centrifugal separation. The obtainedprecipitate was dissolved in a small amount of 20 mM phosphate buffer(pH 7.0), to thereby prepare a crude enzyme solution.

Example 4

Method of defructosylating fructosyl peptide

(Use of defructosylation enzyme originating from a plant belonging tothe family Rosaceae)

(i) A 100 mM acetate buffer (pH 6.0) (100 μL), a 500 μM aqueous solution(40 μL) of one of the N-terminal-valine fructosylated peptides having anamino acid sequence represented by SEQ ID NOs: 1 to 5 (f-VH to f-VHLTPE:products of Bioquest), purified water (20 μL), and the Pyruspyrifolia-origin crude enzyme solution (40 μL) prepared in Example 1were mixed together, and the mixture was caused to react for 64 hours at37° C. The reaction mixture was subjected to ultrafiltration (molecularweight: 10,000), and the filtrate was collected (reaction mixtures 1 to5, respectively) . Each of the reaction mixtures 1 to 5 was analyzedthrough use of a capillary electrophoresis apparatus CAPI-3200 (productof Otsuka Electronics Co., Ltd.) (electrophoresis buffer: 150 mMphosphate buffer (pH 2.0), voltage: 15 kV, detection wavelength: 210 nm)in terms of peak position and peak area.

(ii) Control test

As a control, purified water was added instead of the crude enzymesolution, and the resultant mixture was allowed to react under similarconditions, to thereby prepare a filtrate (control solutions 1 to 5,respectively) . The analysis results obtained from the control solutions1 to 5 were compared with those of the reaction mixtures 1 to 5,respectively.

Each of the fructosyl peptides employed in the enzyme reaction orcontrol test had been mixed with corresponding non-fructosylatedpeptide. The presence or absence of the enzyme activity was determinedthrough comparison of the two peaks.

FIG. 1 shows the results obtained from the reaction mixture 1, and FIG.2 shows the results obtained from the control solution 1. Whereas FIG. 2reveals a peak attributed to f-VH (area: 13 mABU×sec) and a peakattributed to VH (area: 34 mABU×sec), FIG. 1 reveals that the peakattributed to f-VH is lowered (area: 4 mABU×sec) and the peak attributedto VH is increased (area: 38 mABU×sec).

FIG. 3 shows the results obtained from the reaction mixture 2, and FIG.4 shows the results obtained from the control solution 2. Whereas FIG. 4reveals a peak attributed to f-VHL (area: 32 mABU×sec) and a peakattributed to VHL (area: 22 mABU×sec), FIG. 3 reveals that the peakattributed to f-VHL is lowered (area: 7 mABU×sec) and the peakattributed to VHL is increased (area: 34 mABU×sec).

FIG. 5 shows the results obtained from the reaction mixture 3, and FIG.6 shows the results obtained from the control solution 3. Whereas FIG. 6reveals a peak attributed to f-VHLT (area: 38 mABU×sec) and a peakattributed to VHLT (area: 20 mABU×sec), FIG. 5 reveals that the peakattributed to f-VHLT is lowered (area: 5 mABU×sec) and the peakattributed to VHLT is increased (area: 32 mABU×sec).

FIG. 7 shows the results obtained from the reaction mixture 4, and FIG.8 shows the results obtained from the control solution 4. Whereas FIG. 8reveals a peak attributed to f-VHLTP (area: 64 mABU×sec) and a peakattributed to VHLTP (area: 23 mABU×sec), FIG. 7 reveals that the peakattributed to f-VHLTP is lowered (area: 8 mABU×sec) and the peakattributed to VHLTP is increased (area: 57 mABU×sec).

FIG. 9 shows the results obtained from the reaction mixture 5, and FIG.10 shows the results obtained from the control solution 5. Whereas FIG.10 reveals a peak attributed to f-VHLTPE (area: 54 mABU×sec) and a peakattributed to VHLTPE (area: 21 mABU×sec), FIG. 9 reveals that the peakattributed to f-VHLTPE is lowered (area: 9 mABU×sec) and the peakattributed to VHLTPE is increased (area: 48 mABU×sec).

These results substantiate that use of the enzyme originating from aplant belonging to the family Rosaceae is effective for defructosylationof fructosyl peptides.

Example 5

Method of defructosylating fructosyl peptide

(Use of defructosylation enzyme originating from a plant belonging tothe family Vitaceae)

The crude enzyme solution prepared in Example 2 was also tested underconditions similar to those of Example 4. However, as theN-terminal-valine fructosylated peptide, f-VH containing no VH wasemployed, and reaction was performed for 16 hours at 37° C. (reactionmixture 6). A control test was performed in a manner similar to thatdescribed in Example 4 (control solution 6).

FIG. 11 shows the results obtained from the reaction mixture 6, and FIG.12 shows the results obtained from the control solution 6. Whereas FIG.12 reveals only a peak attributed to f-VH (area: 42 mABU×sec), FIG. 11reveals a lowered peak attributed to f-VH (area: 36 mABU×sec) and anewly generated peak attributed to the reaction product (area: 4mABU×sec). In order to identify the new peak, a small amount of VH wasadded to the reaction mixture, followed by capillary electrophoresis.The peak attributed to the reaction product coincided with that of VH,thus confirming the reaction product to be VH.

These results substantiate that use of the enzyme originating from aplant belonging to the family Vitaceae is effective for defructosylationof fructosyl peptides.

Example 6

Method of defructosylating fructosyl peptide

(Use of defructosylation enzyme originating from a plant belonging tothe family Umbelliferae)

The crude enzyme solution prepared in Example 3 was also tested underconditions similar to those of Example 5 (reaction mixture 7).

FIG. 13 shows the results obtained from the reaction mixture 7. For theresults from the control solution, Fig. 12 is again referred to. WhereasFIG. 12 reveals only a peak attributed to f-VH (area: 42 mABU×sec), FIG.13 reveals a lowered peak attributed to f-VH (area: 16 mABU×sec) and anewly generated peak attributed to the reaction product (area: 16mABU×sec) . In order to identify the new peak, a small amount of VH wasadded to the reaction mixture, followed by capillary electrophoresis.The peak attributed to the reaction product coincided with that of VH,thus confirming the reaction product to be VH.

These results substantiate that use of the enzyme originating from aplant belonging to the family Umbelliferae is effective fordefructosylation of fructosyl peptides.

1. A method of defructosylating a fructosylated peptide or protein,comprising reacting the fructosylated peptide or protein with an enzymewhich is extracted from a plant and exhibits defructosylation action. 2.The defructosylation method according to claim 1, wherein the enzymewhich is extracted from a plant and exhibits defructosylation action isextracted from a plant belonging to the family Rosaceae, Vitaceae, orUmbelliferae.
 3. The defructosylation method according to claim 1,wherein the fructosylated peptide has an amino acid sequence representedby any of SEQ ID NOs: 1 to
 5. 4. The defructosylation method accordingto claim 1, wherein the fructosylated protein is hemoglobin Alc.
 5. Anenzyme which exhibits defructosylation action on a fructosylated peptideor protein and is extracted from a plant.
 6. The enzyme according toclaim 5, wherein the plant belongs to the family Rosaceae, Vitaceae, orUmbelliferae.
 7. A method for measuring a fructosylated peptide orprotein, comprising measuring at least one reaction product producedthrough the defructosylation method according to claim
 1. 8. The methodfor measuring a fructosylated peptide or protein according to claim 7,wherein the reaction product produced through the defructosylationmethod is hydrogen peroxide, glucosone, glucose, or a defructosylpeptide.